Method of screening for pharmaceuticals by detecting cross talk between intracellular signals and intranuclear receptors

ABSTRACT

Under the knowledge which two kinds of intracellular signal transfer mechanism of TGF-β super family receptor exist: a pathway via Smad molecule and a pathway via novel MAP kinase such as TAB1 and TAKI, and the knowledge of interaction of Smad molecule with intranuclear receptor or CBP/p300 on the transcription control by Smad molecule, a screening method and a molecular designing method for pharmaceuticals with identified site of action and higher specificity can be accomplished.

BACKGROUND OF THE INVENTION

1. Field of the invention

This invention relates to a method for screening and molecular designingpharmaceuticals useful for various diseases on bone, cartilage, liver,kidney, skin, immune system, central system and the like, or benigntumor, malignant tumor, hyperplasia, fibrosis and the like.

2. Related Background Art

It has been attempted to develop TGF-β super family molecule itself, orantagonist or agonist thereof as a pharmaceutical based on focusing thefunction of TGF-β super family, and some pharmaceutical screeningmethods therefor have been proposed.

For example, (1) as a method for screening by using cell system, thereare screening methods for agonist or antagonist by using the activity ofTGF-β super family in growing, differentiating, killing or maintaininganimal cells as an indicator. Those methods are, for example, (i) ascreening method using inhibition activity of TGF-β on the growth of atumor cell, or propagation accelerating activity of it on the growth ofanother tumor cell as an indicator, (ii) a screening method focusing onthe immuno suppression activity of TGF-β and (iii) a screening methodfocusing on production accelerating effect on extracellular matrix byTGF-β. Furthermore, (2) a screening method focusing on the interactionbetween TGF-β super family and TGF-β receptor super family is known.

However, it is known that TGF-β super family has significantlywide-ranging functions, and thus, a mere agonist or antagonist for TGF-βsuper family may have a problem such that main function and sidefunction can not be sufficiently differentiated, and consequently theyare not sufficient for a screeningmethod for pharmaceuticals. That is,the above screening method can not differentiate only main function fromother functions, and very likely screen materials with low specificitynot to be an candidate for pharmaceuticals. Furthermore, even if auseful candidate for pharmaceuticals could be screened, it still remainsdifficulty to find guideline for synthesizing a useful derivatives andto do molecular designing.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a novel method forscreening pharmaceuticals, which overcomes the above prior art'sproblems.

As a result of intensive efforts to overcome the above-mentionedproblems, the present inventor succeeded to develop a screening methodfor pharmaceuticals (inhibitor, accelerator etc.) with more specificityand a method for molecular designing them, based on a novel principle byfocusing on intracellular signal transfer mechanism of TGF-β receptorsuper family, and accomplished this invention.

Although a pathway via Smad molecule and the MAP kinase pathway viaTAB1, TAK1 and the like have been known as an intracellular transfermechanism of TGF-β receptor super family, it is little understood howthe expression of the targeted gene is finally controlled by thesepathways. Especially, it is known that Smad molecule affects totranscription control in nucleus, but a transcription control factorinteracted with Smad molecule in higher animal cell has not beendetermined.

The present inventor demonstrated that the interaction among Smadmolecule, intranuclear receptor and transcription coupling factor isimportant physiologically in the transcription control mechanism of Smadmolecule. Under the above-mentioned knowledge, the present inventordeveloped a method for screening and a method for molecular designingpharmaceuticals based on the novel principle which focuses on thetranscription control mechanism via the intracellular signal transfermechanism of TGF-β receptor super family.

In the signal transfer pathway of Smad, as a pharmacofunction beingpresumable from the prior art, there are (i) phosphorylation into Smad2, 3 and the like (obtaining transition ability into nucleus), (ii)dimer formation of Smad molecule, and (iii) discovering of inhibitoragainst the signal transfer pathway such as Smad 6, 7, or controlling offunction of the pathway. However, because these pharmacofunctions areappeared in cytoplasm but not in ucleus and the pathway is wellconserved, a screening method for pharmaceuticals based on suchfunctions is not suitable to search pharmaceuticals with highspecificity. Furthermore, other than the above (ii), the concreteinteraction between molecules has not been clarified, and thus atheoretical approach for molecular designing based on the structure isnot appropriate.

On the other hand, in connection with the transcription control by Smadmolecule, the present inventor obtained the knowledge about theinteraction of Smad molecule with intranuclear receptor or CBP/p300,thereby clarified a tangible partner of Smad. According to suchknowledge, the present inventor developed a screening method forpharmaceuticals with specified site of action and high specificity. Byusing the above method, it becomes possible to find usefulpharmaceutical candidates and to provide a guideline or moleculardesigning for synthesizing more useful materials. Based on suchknowledge, pharmaceutical designing becomes possible. Various kinds ofknown techniques to determine the three dimensional structure such asX-ray crystallographic structure analysis, spectroscopic analysis suchas NMR (nuclear magnetic resonance), IR(Infrared), and CD(circulardichroism), or various biological modification such as a point mutationtechniques can reveal the three dimensional structure of the bindingsite of a Smad molecule and intranulcear receptor and transcriptioncoupling factor. Thus obtained three dimensional structural data can beused to design pharmaceuticals, if necessary with a numericalcalculating method such as a commercially available molecular modelingcomputer softwares (SYBYL(Tripos Inc.,St.Louis,USA);InsightII/Discover(Molecular Simulations Inc., San Diego,USA);Quanta/CHARMM(Molecular Simulations Inc., San Diego,USA);LOOK(Molecular Applications Group, Palo Alto, USA);CAChe(OxfordMolecular Group, Oxford, UK).

Furthermore, base on the above principle, it becomes possible to developa screening method for an agonist or an antagonist of intranuclearreceptor (for example, vitamin D receptor) with higher specificity.Since intranuclear receptors are distributed over various tissues, it isimpossible to avoid some side effect of agonist or antagonist typepharmaceuticals screened by the prior method, which such intranuclearreceptors are simply targeted. However, upon focusing on the interactionbetween Smad molecule and intranuclear receptors, according to thepresent invention, it is possible to inhibit or accelerate only thespecific transcription control using Smad as a transcription couplingfactor. Therefore, it becomes possible to search excellentpharmaceuticals with good efficiency and specificity (targeting variousdisease of bone, skin, liver, kidney, immune system, center system andthe like, or benign tumor, malignant tumor, hyperplasia or fibrolysis)in addition to the known usefulness of intranuclear receptor agonist orantagonist.

In other words, the present invention provides a method to develop anovel therapeutic agent based on the novel knowledge of the interactionof Smad molecule with intranuclear receptor or with CBP/p300.

The present invention will be more fully understood from the detaileddescription given hereinbelow and the accompanying drawings, which aregiven by way of illustration only, and thus are not to be considered aslimiting the present invention.

Further scope of applicability of the present invention will becomeapparent from the detailed description given hereinafter. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the results of Example 1.

FIG. 2 is a diagram showing the results of Example 2.

FIG. 3 is electrophoresis patterns showing the results of Example 3.

FIG. 4 is a diagram showing the results of Example 4.

FIG. 5 is a diagram showing the results of Example 4.

FIG. 6A is electrophoresis patterns showing the results of Example 3 inthe case of p300.

FIG. 6B is electrophoresis patterns showing the results of Example 3 inthe case of CBP.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention will be hereinafter explained in detail along with someembodiments.

A battery of factors belonging to TGF-β super family such as TGF-β andBMP show various physiological functions and they act as aggravationfactor or amelioration factor invariousdiseaseofbone, cartilage, liver,kidney, skin, immune system and the like or carcinoma. On the otherhand, functions via vitamin D3, retinoic acid, steroid hormone andreceptors thereof (intranuclear receptor/steroid hormone receptor superfamily) are also various, and target tissues thereof are overlappedthose of the above TGFβ super family.

The present inventor found that the intracellular signal of TGF-βaffects the transcription control of intranuclear receptors, that suchfunction is expressed via intracellular signal transfer molecule ofTGF-β, Smad, and that Smad3 affects particularly to transcriptionactivity of VDR as transcription coupling factor and intermediates theinteraction between VDR and CBP/p300.

Furthermore, the present inventor found that Smad3 directly binds toCBP/p300 which is thought to be indispensable to activation of almostall intranuclear receptors, and thereby elucidated the cross-talk atmolecular level between TGF-β intracellular signal and wide-rangingintranuclear receptor transcription control.

Upon elucidation of the cross-talk between TGF-β intracellular signaland intranuclear receptor transcription control, an entirely differentapproach to create therapeutic agent involving TGF-β and intranuclearreceptor, especially VDR becomes possible.

In other words, a pharmaceutical agent affecting intracellular signal ofTGF-β and intranuclear receptor transcription activity improves tissuespecificity and efficiency, which have not been accomplished heretofore.And the pharmaceutical agent enables to establish various kind ofscreening system based on molecular interaction. Thus, materials foundby the screening method based on such knowledge include effectivetherapeutic agents for diseases of bone, cartilage, liver, kidney, skin,immune system and central system, or benign tumor or malignant tumor,hyperplasy, fibrosis and the like.

While TGF-β employs Smad2 or 3 and Smad4 for intracellular signaltransfer, BMP belonging to TGF-β super family employs Smadl and Smad4having similar structure for intracellular signal transfer. The presentinvention provides a novel screening method using mainly TGF-β, Smad3,CBP/p300 and various intracellualr receptors, but it can be easilyassumed to be common to BMP and Smadl.

Because Smad3 binds to CBP/p300 directly, this invention is alsoapplicable to screening of transcription activity control materials oftranscription activating factors (CREBP, AP-1 and the like) other thanintranuclear receptors.

The screening method of this invention will be exemplified.

Screening Method (I):

Based on the knowledge found by the present inventor, it is possible toscreen cross talk between intracellular signal from TGF-β familyreceptors and intranuclear receptors and to screen (search) materialsaffecting the cross talk (see Example 1).

Screening Method (II):

A screening method is possible by using the Two-Hybrid System(Gyuris,J., Cell, 1993,75:791-803; Golemis, E. A., Current Protocols inMolecular Biology [John Wiley & Sons, Inc.], 1996, Ch. 20.0 and 20.1).In order to screen materials affecting the interaction (binding) betweenintranuclear receptor and Smad molecule, the Two-Hybrid System can beused.

As a transcription activating domain used for the Two-Hybrid System,forexample, GAL4 transcription activating domain (Brent, R., Cell,1985, 43:729-736), [Bicoid], [c-Fos], [c-myc], [v-Myc], [B6], [B7], [B42](Golemis, A. E., Mol. Cell Biol., 1992,12:3006-3014) or [VP16] (CLONTECHCorp., Mammalian MATCHMAKER Two-Hybrid Assay Kit) may be listed.

A DNA binding domain used for the Two-Hybrid System, for example isidentified in transcription factors such as GAL4 (Giniger, E., Cell,1985,40: 767-774) p53(Chumakov, P. M.,Genetika, 1988,24:602-612), GCN4(Hinnenbush, A. G., Proc. Natl. Acad. Sci., 1984, 81: 6442-6446), VP16(Triezeneberg, S. J., Genes. Dev., 1988, 2: 718-729) ReloA (Nolan, G.P., Cell, 1991, 64: 961-969) Oct-1 (Strum, R. A., Genes. Dev., 1988, 2:1582-1599) c-Myc (Watt, R., Nature, 1983, 303:725-728), c-Jun (Angel,P., Cell, 1988, 55: 875-885), MyoD (Write, W. E., Cell, 1989, 56:607-617).

The principle to screen materials affecting the binding between proteinsA and B by using the Two-Hybrid System is explained hereinbelow. Anexpression vector for the fusion protein of the above-mentionedtranscription activating domain and the protein A, an expression vectorfor the fusion protein of the above-mentioned DNA binding domain and theprotein B, and an eucaryotic host having an expression unit comprisingan appropriate reporter gene in its nucleus are prepared. The reportergene is constructed as an expression unit consisting of promoter regionwhich is activated when both fusion proteins bind together and thereporter gene linked to the promoter region at the downstream of theregion.

It is possible to screen materials affecting the binding of proteins Aand B by detecting the expression of the reporter gene and selecting amaterial affecting the amount of the expression.

An example of the expression vector may be used any expression vectorwithout any restriction as long as it can stably express proteins codedby (1) fusion DNA fused with DNA coding the above-mentionedtranscription activating domain and an intranuclear receptors or Smad,and (2) fusion DNA fused with DNA coding the above-mentioned DNA bindingdomain and an intranuclear receptor or Smad in eucaryote hosts. Anexample of intranuclear receptor is preferably VDR, but otherintranuclear receptor/hormone receptors, such as RARAα, β, γ, RXRα, β,γ, PPARα, β, γ, ER, AR, PgR, GR and the like may be used without anyrestriction. Any DNA coding an intranuclear receptor may be used as longas it includes DNA coding ligand/hormone binding domain. An example ofSmad molecule is preferably Smad3, but DNA coding Smad2 or othermolecules belonging to Smad family may be used without any restriction.

Any expression vector for reporter gene may be used as long as itincludes the promoter region activated upon the binding of the bothfusion proteins and reporter gene linked to the promoter region at thedownstream of the gene and can express the reporter gene productsspecifically and efficiently when the both fusion proteins bind together(see Example 2).

Screening Method (III):

A screening method is possible by employing antigen antibody reaction.After Smad molecules and intranuclear receptors are expressed in cellsand those cells are cultivated with a material to be tested for acertain period, cells are crushed to prepare cell lysate. Effect of thematerial to be tested against the interaction (binding) of bothmolecules (Smad-intranuclear receptor) can be detected by immuneprecipitation with antibody against one of those molecules, anddetecting or quantifying another molecule involved in the immuneprecipitate with a immunological technique. The screening for materialsinducing the binding of the both molecules is easily accomplished byadding a material to be tested alone to the above-mentioned culturesystem and performing the above-mentioned experiments, and thencomparing to the immune precipitate obtained from cells without thematerial. Material modifying (accelerating or suppressing) or inhibitingthe binding of the both molecules can be screened by adding anappropriate agonist/hormone of an intranuclear receptor and a materialto be tested to the above-mentioned culture system simultaneously andperforming the above described experiments, and then by comparing toimmune precipitate obtained from cells without the material.

Smad and intranuclear receptors may be used by expressing as a fusionprotein fused with tag such as FLAG-, HA-, His-, Fc portion ofimmunoglobulin, GST-, GFP or labeled protein/ peptide. In such case, anantibody used for immune precipitate or immunological detection may bean antibody recognizing those tags. Instead of immune precipitation withan antibody, the targeted protein complex can be captured by using Ni orglutathione immobilized on a solid surface such as beads. Furthermore,in order to detect the targeted protein, by using the feature of thefusion tag/labeled protein/peptide, it is possible to detect thetargeted protein based on its enzyme activity and fluorescence activity.As the intranuclear receptor, a ligand binding domain alone, or anyfusion protein including a ligand binding domain can be used.

When the targeted protein in the Smad—intranuclear receptor complexobtained by immune precipitate or the above-mentioned method is to bedetected or quantified, separation by SDS-polyacrylamide gelelectrophoresis and detection by a western blotting can be used. ELISAor RIA can be also used to quantify it.

As a simple laboratory procedure, it is possible to immobilize anantibody which recognizes one of the fusion protein molecules or thefusion protein itself, Ni, glutathione and the like on ELISA plate orbeads, to capture Smad—intranuclear receptor complex from theabove-mentioned cell lysate, and to detect or quantify the targetedprotein molecule in the captured complex by using an antibodyrecognizing another protein molecule. For example, like commonly used indetection procedures, a direct assay using an antibody labeled with anenzyme or radioisotope, an indirect assay using labeled secondaryantibody, or an biotin-avidin assay which uses biotin-labeled antibodyand quantifies with labeled avidin can be used. Where one of the proteinmolecules is expressed as a fusion protein with fluorescenceproteinsuchas GFP, the interaction (binding condition) of Smad andintranuclear receptor can be determined by measuring the fluorescencedirectly after capturing Smad—intranuclear complex on solid surface withfor example an antibody recognizing another protein molecule (seeExample 3).

Screening Method (IV):

Based on the interaction of Smad and intranuclear receptors, materialsaffecting the interaction can be screened in cell-free system (seeExample 4).

Screening method (V):

A screening can be done by detecting the interaction of transcriptioncoupling factor and Smad molecule. CBP and P300 act as a transcriptioncoupling factor mediating transcription activity from many transcriptioncontrol factors including intranuclear receptors. VDR does not bindCBP/P300 directly. However it is demonstrated that VDR binds CBP/P300via Smad3 as described below.

Consequently, it is necessary to sufficiently consider functions overthe binding of Smad molecule and CBP/P300 in order to screen materialsaffecting the transcription control by Smad molecule and intranuclearreceptor. Thus, a screening system for materials affecting the bindingSmad molecule and CBP/p300 can be established by applying the followingexamples.

The experimental result using dominant negative Smad3 suggests thatSmad3 accelerates transcription control by cAMP, phorbol ester or PDGF(Istvan Mucsi, et al., BBRC, 232, 517-521, 1997). However, since themechanism has not been demonstrated, it has been difficult to screenmaterials modifying such wide-ranging functions of Smad3. The presentinvention makes such wide-ranging functions via Smad3 understandablesystematically as the interaction of Smad molecule and CBP/p300.Consequently, it becomes possible to develop a screening system formaterials affecting the binding of the above-mentioned both molecules.

An example of such screening method is described below. The method usesGST-pull down method (described supra) employing GST-Smad fusionmolecule and labeled CBP/p300, and can detect materials modifying thebinding of the both molecules by adding a material to the reactionsystem upon reaction of the both molecules. Also, GST-pull down methodemploying GST-CBP and GST-p300 fusion protein and labeled Smad moleculecan be used.

Any system which can measure the interaction (binding) between proteinsother than GST-pull down method described above enables to screenmaterials accelerating or suppressing the interaction of Smad moleculeand CBP/p300. Such system includes both cell and cell-free system, andcan use easily ELISA, RIA, SPA method, BIACORETM, fluorescencepolarization method, and the Two-Hybrid System. A partial peptide ofSmad molecule or CBP/p300 molecule can be used. Such partial peptide ofSmad or CBP/p300 molecule can be used as a fusion protein fused with tagsuch as FLAG-, HA-, His-, Fc portion of immunoglobulin, GST-, GFP orlabeled protein/peptide.

EXAMPLE

This invention will be hereinbelow explained in detail by some examples.However, this invention is not limited by the following examples. Thecontents of Application No.10-17818, filed Jan. 14, 1998, in Japan ishereby incorporated by reference.

Example 1

TbetaR-I TD in which a gene coding constructively active modified TGF-βtype I receptor (TβRI-TD) was introduce into higher animal expressionvector was used to introduce TGF-β signal into cells constructively.

The gene coding androgen receptor (AR), the gene coding vitamin D3receptor (VDR), or the gene coding retinoic acid receptor (RAR) wereintroduced into higher animal expression vector pSG5, respectively, andeach expression plasmids for respective receptors was constructed(pSG5-AR, pSG5-VD or pSG5-RAR), and those plasmids were used in thefollowing experiments. Plasmid with only each expression vector was usedas control.

Reporter gene expression plasmid was used in which the sequencecorresponding to each hormone receptor/intranuclear receptor wasinserted into CAT reporter gene expression vector.

COS-1 cells which were cultured in Dulbeccols modified Eagle's medium-5%dextran-coated activated charcoal treated fetal calf serum withoutphenol red were used for the following experiments. COS-1 cells werecultivated in a 10 cm diameter Petri dish. At the point that cellssaturation density was reached to 40-50%, the cells were combined withabove-mentioned plasmids according to each purpose to make 20 μg intotal, and the plasmids were introduced into cells by calciumcoprecipitation method. As a internal reference, 3 μg of the expressionvector pCH110 (Pharmacia) of β-galactosidase gene was introduced intocells simultaneously, and the introduction efficiency among respectivePetri dishes was adjusted based on the amount of expression of the gene.Each matrial to be tested such as testosterone, 1,25(OH)2VD3 andretinoic acid was added into Petri dish one hour after the geneintroduction, and thereafter was also added upon medium exchange.

Twenty-four hours after the gene introduction, cells were washed withfresh culture medium and continued to be cultured for additional 24hours. Cells were crushed with freeze and thaw method to prepare cellextract. CAT activity was measured and concurrently β-garactosidaseactivity were measured to adjust the data.

The result showed that the transcription activity made by the additionof testosterone or retinoic acid via AR or RAR was slightly reinforcedby signal from TGF-β. The transcription activity made by the addition of1,25(OH)2VD3 via VDR was significantly reinforced by signal from TGF-β(FIG. 1). These results demonstrate that intracellular signal of TGF-βaffects (reinforces) transcription activity of hormonereceptor/intranuclear receptor.

Accordingly, by using this analysis system, it is possible to screenmaterials affecting transcription control mechanism of hormonereceptor/intranuclear receptor mediated by intracellular signal ofTGF-β.

In other words, it is possible to screen hormone agonist or antagonistwhich have different dependency on TGF-β signal—intranuclear receptorinteraction by the method which comprises addition of any variousmaterial to be tested into the system instead of physiological ligandssuch as testosterone, 1,25(OH)2VD3 or retinoic acid, or together withthem in the presence or absence of signal of TGF-β and comparison thetranscription activity of each receptor. It is likewise possible toscreen accelerator or inhibitor of TGF-β signal—intranuclearinteraction.

Example 2 Two-hybrid analysis using higher animal cells

COS-1 cells which were cultured in Dulbecco's modified Eagle's medium—5%dextran-coated activated charcoal treated fetal calf serum withoutphenol red were used for the following experiments. COS-1 cells werecultivated in a 10 cm diameter Petri dish. At the point that cellsaturation density was reached to 40-50%, 20 μg of the following plasmidin total was introduced into cells by calcium coprecipitation method. Asa internal reference, 3 μg of the expression vector pCH110 (Pharmacia)of β-galactosidase gene was introduced simultaneously, and introductionefficiency among respective Petri dishes was adjusted based on theamount of the expression of the gene. Each material to be tested such asVD3 was added into Petri dish one hour after the gene introduction, andthereafter was also added upon medium exchange. Twenty-four hours afterthe gene introduction, cells were washed with fresh culture medium andcontinued to be cultured for additional 24 hours. Cells were crushedwith freeze and thaw method to prepare cell extract. CAT activity wasmeasured and β-garactosidase activity was measured concurrently toadjust the resulting data.

As a plasmid for the two-hybrid analysis, the gene coding D,E,F domainsof VDR was introduced into pM(Clontech) vector and thereby theexpression plasmid of GAL4 DNA-binding domain—VDR(DEF) fusion proteinwas constructed. Also, genes coding Smad2 and 3 were introduced intopVP16(Clontech) vector and those were used as expression plasmid forVP16—Smad2 or VP16—Smad3 fusion proteins. As a reporter gene expressionplasmid, 17M5CAT in which CAT gene was inserted into 17M5 (including 5times of USA 17 mer of GAL-4 recognition site). These expressionplasmids were introduced into COS-1 cells as described in above.

In cells which expressed GAL4 DNA-binding domain—VDR(DEF) andVP-16-Smad2 or GAL4 DNA-binding domain—VDR(DEF) and VP-16-Smad3,stronger CAT activity increase was detected in the presence of 1 nM of1α, 25(OH)2VD3, compared with that in the absence of 1α, 25(OH)2VD3 (seeFIG. 2). It was shown that Smad3 and VDR bonds each other in thepresence of 1α, 25(OH)2VD3.

By using this analysis system, it is possible to screen materialsreinforcing the interaction of Smad3 and VDR. It is also possible toscreen materials inhibiting the binding of Smad2 or Smad3 and VDRrecognized in the presence of VD3 agonist such as 1,25(OH)2VD3.

Example 3

Immune Precipitate Method—immunoblotting Method

The following two kinds of expression plasmids were introduced intoCOS-1 cells by the same procedure as set forth Example 1. One hour afterthe gene introduction, 1,25(OH)2VD3 was added into Petri dish to be 1 nMat its concentration or not added (for comparison), and thereafter wasalso added at same concentration upon medium exchange. Twenty-four hoursafter the gene introduction, cells were washed with fresh culture mediumand continued to be cultured for additional 24 hours. (1) pSG5-VDR inwhich VDR gene is inserted into higher animal expression vector pSG5,and (2) the plasmid in which FLAG-tagged Smad3 gene is inserted intohigher animal expression vector pcDNA3 (Invitrogen) were used. Afterlysing cells, immune precipitation was done with anti-FLAG monoclonalantibody M2 (IBI; Eastman Kodak). Then, coprecipitated proteins wereseparated by 8% SDS-polyacrylamide gel electrophoresis. Separatedproteins were transferred onto PVDF membrane (Bio-Rad), and then VDR wasdetected by using rabbit anti VDR antibody and alkaline phosphataselabeled anti rabbit IgG antibody (Promega).

The results show that VDR was barely detected in the example using cellswithout 1,25(OH)2VD3. On the other hand, the VDR band was clearlydetected in the example using cells added with 1,25(OH)2VD3 (FIG. 3).

By using this analysis system, it is possible to screen materialsaccelerating the interaction (binding) of Smad3 and VDR. Furthermore, itis possible to screen materials inhibiting the interaction (binding) ofSmad3 and VDR in the presence of agonist such as 1,25(OH)2VD3 by addingany materials into this culture system.

Example 4

Detection System for Interaction of CDR and Smad Molecule

—GST-pull Down Analysis—

GST fused Smad3 or VDR (full length peptide 1-424, partial peptides:amino acid sequence 115-424, 115-357, 115-300 and others, see FIG. 4)fused proteins (GST-Smad3 or GST-VDR) were expressed by using E.coli,and were captured on beads with glutatione-Sepharose bead (Pharmacia) toaccomplish affinity purification. On the other hand,³⁵S-methionine-labeled Smad3 and VDR were prepared in cell-free systemby using reticulocyte lysate translation system (Promega). GST-Smad3 orGST-VDR fixed on beads described above was incubated with ³⁵S-methioninelabeled VRD or Smad3. By using polyacrylamide gel electrophoresis,³⁵S-methionine labeled VDR or Smad3 were analyzed.

The results show that VDR partial peptides, 115-357 and 115-300 bound toSmad3. This binding did not require 1,25(OH)2VD3. On the other hand,full length VDR slightly bound to Smad3 in the presence of 1 nM of1,25(OH)2VD3 (FIG. 5).

The disjunction from the result of 1,25(OH)2VD3 in cell system ispresumed that in cell system, in the presence of 1,25(OH)2VD3,intranuclear receptor transcription coupling factor (SRC-1/TIFII familyprotein) interacts with AF-2 core domain located near to C-terminal ofVDR to result in the structure change of VDR and to make partial peptideof main central portion of VDRE domain (amino acid: 115-357, 115-300)possible to interact with (bind to) Smad molecule. In cell-free system,the strong binding of full length VDR and Smad molecule is not detectedeven though 1,25(OH)2VD3 is added into the reaction system because oflacking transcription coupling factor.

Because VDR(115-300) and VDR(115-357) are VDR partial peptidescontaining binding site with Smad3, it is possible to screen materialsaccelerating or inhibiting VDR—Smad interaction (binding) by adding amaterial to be tested into GST-pull down analysis system usingGST-VDR(115-300) or GST-VDR(115-357) and labeled Smad3.

Any analysis system other than GST-pull down method, in whichinteraction (binding) between proteins can be measured enables to screenmaterials accerelating or inhibiting the interaction of Smad moleculeand intranuclear molecule. Such measurement systems are possibly ELISA,RIA, SPA method, BIACORE™, fluorescence ellipsometric method.Furthermore, respective partial peptides of Smad molecule andintranuclear receptor molecule can be used.

Moreover, respective partial peptides of Smad or intranuclear receptormolecule can be used as a fusion protein fused with tag such as FLAG-,HA-, His-, Fc portion of immunoglobulin, GST-, GFP or labeledprotein/peptide.

Example 5

Detection for interaction of Smad molecule and CBP/p300

—GST-pull down method—

GST fused Smad3 fusion protein (GST-Smad3) was expressed by usingE.coli, and was captured on beads with glutatione-Sepharose bead(Pharmacia) to accomplish affinity purification. On the other hand,³⁵S-methionine-labeled CBP and p300 were prepared in cell-free system byusing reticulocyte lysate translation system (Promega). GST-Smad3 fixedon beads described above was incubated with ³⁵S-methionine labeled CBPor p300. After accomplishing SDS-polyacrylamide gel electrophoresis,³⁵S-methionine labeled CBP or p300 was analyzed by usingautoradiography. The results show that CBP and p300 bound to Smad3directly (FIGS. 6A and 6B).

In this analysis system, when GST-Smad3 and ³⁵S-methionine labeled CBP(GST-Smad3 and ³⁵S-methlonine labeled p300) are reacted together, theresultant autoradiographical band of CBP or p300 would be increased ordecreased in the presence of any material accelerating or inhibiting thebinding of these molecules. By using such change as indicator, it ispossible to screen materials modifying (accelerating or inhibiting) theinteraction of Smad3 and CBP/p300.

As a summary, based on the novel knowledge which intracellular signaltransfer mechanism from TGF-β super family by TGF-β super familystimulation do cross talk with intranuclear receptor or transcriptioncontrol factor CBP/p300 via Smad molecule, a screening method forpharmaceuticals (such as inhibitor, accelerator) with high specificitycan be accomplished.

From the invention thus described, it will be obvious that the inventionmay be varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedfor inclusion within the scope of the following claims.

What is claimed is:
 1. A method of screening for a substance thatinhibits or induces interaction between an intranuclear receptor and aSmad molecule comprising the following steps: a. preparing a culturesystem consisting of introducing an intranuclear receptor expressionvector and a Smad molecule expression vector into cultured cells andculturing said cells; b. adding a substance to be tested to said culturesystem; and c. measuring the inhibition activity or induction activityof said substance by detecting the binding of said intranuclear receptorto said Smad molecule.
 2. A method of screening for a substance asclaimed in claim 1, wherein said binding of said intranuclear receptorto said Smad molecule is detected by the Two-Hybrid System.
 3. A methodof screening for a substance as claimed in claim 1, wherein said bindingof said intranuclear receptor to said Smad molecule is detected by anantigen-antibody reaction.
 4. A method of screening for a substance asclaimed in claim 1, wherein said intranuclear receptor is a vitamin Dreceptor.
 5. A method of screening for a substance as claimed in claim1, wherein said Smad molecule is Smad3.
 6. A method of screening for asubstance that inhibits or induces interaction between an intranuclearreceptor and a Smad molecule, by detecting interaction between anintranuclear receptor and a Smad molecule comprising: physicallycontacting said intranuclear receptor with said Smad molecule in thepresence or absence of a substance to be tested; detecting the bindingof said intranuclear receptor to said Smad molecule; and measuring theinhibition activity or induction activity of said substance.
 7. A methodof screening for a substance as claimed in claim 6, wherein said bindingof said intranuclear receptor to a Smad molecule is detected by GST-pulldown analysis.
 8. A method of screening for a substance as claimed inclaim 6, wherein said binding of said intranuclear receptor to a Smadmolecule is detected by a method selected from the group consisting ofELISA, RIA, SPA, BIACORE™ and fluorescence polarization methods.